Re: why does high NA excitation illumination give better resolution in fluorescence microscopy?

Posted by Arne Seitz on
URL: http://confocal-microscopy-list.275.s1.nabble.com/why-does-high-NA-excitation-illumination-give-better-resolution-in-fluorescence-microscopy-tp7581519p7581521.html

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Hi Dan,

the discussions so far are strictly true for wide field microscopy. There the NA of the condenser should not play a role (given enough SNR). And to my understanding light-sheet microscopy is just a prominent example of such a scenario.
For confocal microscopy things are to my understanding a bit more complicated. The gain in lateral resolution with an infinitesimal small pinhole is due to the fact that the excitation point-spread function is convolved with the emission point spread function. Thus in that case the NA of the condensor should play a role.  But as the lateral resolution improvement is hardly seen in practice this is just a theoretical limitation.

Cheers
Arne

> Hi again all,
>
> So from the informative answers so far...
> it seems that the idea of filling the back focal plane of the objective (acting as
> the condenser lens) in epi fluorescence, both widefield and confocal, is more
> about getting lots of light into the sample in order to get enough signal to
> then be able to see the resolution that is there, since effective resolution is
> limited by signal to nose (sorry, noise)
>
> In other words, is there enough light going in (and consequently coming back
> out) that we can see the Rayleigh criterion dip between the two peaks of the
> object images,  despite any noise that's present?
>
> Is this following statement then true?:
>
> In the case of infinite signal to noise, where contrast is optimal and not
> limited by noise, where illumination power is close to saturation of the
> fluorescence excited state, exposure time is long enough, etc....
> the effective NA of illumination (back aperture filling) has no effect on
> achievable lateral or axial resolution - only signal:noise does.
>
> Are there cases where this is false? Anisotropy?
>
> cheers
>
> Dan